1. Field of the Invention
Mycophenolic acid ("MPA") is produced by the fermentation of several penicillium species. It has a broad spectrum of activities, specific mode of action, and is tolerable in large doses with minimal side effects, Epinette, et al., Journal of the American Academy of Dermatololqy 17(6):962-71 (1987). MPA has been shown to have antitumor, antiviral, antipsoriatic, immunosuppressive, anti-inflammatory activities, Lee, et al., Pharmaceutical Research 7(2):161-166 (1990), along with antibacterial and antifungal activities, Nelson, et al., Journal of Medicinal Chemistry 33(2):833-838 (1990). It inhibits inosine monophosphate dehydrogenase, an enzyme in the de novo synthesis of purine nucleotides. Since T and B lymphocytes depend largely upon this de novo synthesis, MPA is able to inhibit lymphocyte proliferation, which is a major factor of the immune response.
The morpholinoethyl ester of MPA, morpholinoethyl (E)-6-(1,3-dihydro4-hydroxy-6-methoxy-7-methyl-3-oxo-5-isobenzofuranyl)-4- methyl-4-hexenoate ("MPA-M") is rapidly hydrolyzed in vivo to MPA. Administration of MPA in the form of this ester, greatly improves MPA's bioavailability.
Because MPA is a potent biologically active material, an effective immunoassay could be useful in monitoring its bioavailability. In addition, it may be important to monitor therapeutic drug levels, i.e., optimal drug levels necessary for adequate immunosuppression. Since MPA-M is rapidly hydrolyzed to MPA, an assay for MPA would provide means of regulating and optimizing MPA-M dosages.
MPA levels in patient samples have been determined by high performance liquid chromatography (HPLC). Prior to conducting the HPLC analysis, the sample to be determined was subjected to solid phase extraction using organic solvent elution or direct organic solvent extraction.
2. Description of the Related Art
Nowak, et al., Clin. Chem. (1995) 41(7): 1011-1017 discusses mycophenolic acid binding to human serum albumin: characterization and relation to pharmacodynamics.
Langman, et al., Therapeutic Drug Monitoring (1994) 16:802-807 discusses blood distribution of mycophenolic acid.
European Patent 0 218 309 B1 discloses a method for measuring free ligands in biological fluids. Sodium salicylate and 2,4-dinitrophenol were employed to prevent labeled analogs of triiodothyronine and tetraiodothyronine from binding to albumin and thyroid binding pre-albumin.
European Patent Application 0 392 332 A2 discloses a fluorescent polarization immunoassay and reagents therefor. Various compounds were disclosed for converting a marijuana metabolite, which was bound to serum albumin and other proteins in urine, to free form. These compounds included, among others, 8-anilino-1-naphthalenesulfonic acid (ANS), salicylic acid and 5-methoxysalicylic acid.